Pathological characteristics of axons and proteome patterns in midbrain dopaminergic neurodegeneration induced by WDR45-deficiency

Background Although WD repeats domain 45 (WDR45) mutations have been linked to β-propeller protein-associated neurodegeneration (BPAN), the precise molecular and cellular mechanisms behind this disease remain elusive. This study aims to shed light on the effects of WDR45-deficiency on neurodegeneration, specifically axonal degeneration, within the midbrain dopaminergic (DAergic) system. By examining pathological and molecular alterations, we hope to better understand the disease process. Methods To investigate the effects of WDR45 dysfunction on mouse behaviors and DAergic neurons, we developed a mouse model in which WDR45 was conditionally knocked out in midbrain DAergic neurons (WDR45cKO). Through a longitudinal study, we assessed alterations in mouse behavior using open field, rotarod, Y-maze, and 3-chamber social approach tests. To examine the pathological changes in DAergic neuron soma and axons, we utilized a combination of immunofluorescence staining and transmission electron microscopy. Additionally, we performed proteomic analyses of the striatum to identify the molecules and processes involved in striatal pathology. Results Our study of WDR45cKO mice revealed a range of deficits, including impaired motor function, emotional instability, and memory loss, coinciding with the profound loss of midbrain DAergic neurons. Prior to neuronal loss, we observed massive axonal enlargements in both the dorsal and ventral striatum. These enlargements were characterized by the accumulation of extensively fragmented tubular endoplasmic reticulum (ER), a hallmark of axonal degeneration. Additionally, we found that WDR45cKO mice exhibited disrupted autophagic flux. Proteomic analysis of the striatum in these mice showed that many differentially expressed proteins (DEPs) were enriched in amino acid, lipid, and tricarboxylic acid metabolisms. Of note, we observed significant alterations in the expression of genes encoding DEPs that regulate phospholipids catabolic and biosynthetic processes, such as lysophosphatidylcholine acyltransferase 1, ethanolamine-phosphate phospho-lyase, and abhydrolase domain containing 4, N-acyl phospholipase B. These findings suggest a possible link between phospholipid metabolism and striatal axon degeneration. Conclusions In this study, we have uncovered the molecular mechanisms underlying the contribution of WDR45-deficiency to axonal degeneration, revealing intricate relationships between tubular ER dysfunction, phospholipid metabolism, BPAN and other neurodegenerative diseases. These findings significantly advance our understanding of the fundamental molecular mechanisms driving neurodegeneration and may provide a foundation for developing novel, mechanistically-based therapeutic interventions.

Mutations in these ER-shaping proteins can cause hereditary spastic paraplegia (19). The accumulation of tubular ER is primarily speci c to axons (20). Recent studies have revealed the physiological roles of tubular ER in neurodegenerative disorders (19,21,22). Autophagy gene 5 (ATG5) deletion leads to tubular ER accumulation in axons of hippocampal neurons (20), demonstrating that autophagy participates in the axonal tubular ER degradation.
The tubular ER dynamics are closely linked to phospholipid synthesis. Cytidine diphosphatetriacylglycerol (CDP-DAG) synthase 1, a crucial enzyme of PC synthesis through the CDP-DAG pathway, is uniformly localized to the tubular ER and nuclear envelope. Phosphatidylinositol synthase (PIS), the ratelimiting enzyme for phosphatidylinositol synthesis, also localizes to the tubular ER network. Rab10, an ER-speci c Rab GTPase, regulates ER tubule dynamics and tubular ER morphology by controlling ER tubule extension and fusion at the leading edge of ER dynamics. Further study suggests that these dynamics could be coupled to phospholipid synthesis since the Rab10 domain is highly enriched with at least two ER enzymes that regulate phospholipid synthesis, PIS, and choline/ethanolamine phosphotransferase 1 (23).
Using a line of newly developed WDR45 conditional knockout (cKO) mice, our study shows a correlation between axonal degeneration and the accumulation of aberrant tubular ER in WDR45-de cient midbrain DAergic neuron. These ndings suggest that the dysfunction of autophagy and alterations in lipid metabolism, particularly phospholipid metabolism, may play a role in striatal axonal degeneration. By uncovering these connections, our study provides new insights into the molecular mechanisms underlying WDR45-de ciency-induced neurodegeneration and highlights potential targets for developing therapeutic interventions to prevent or reverse this process.

Materials and methods
Generation of the conditional knockout WDR45 Mouse model Heterozygous WDR45 Flox/wt mice were generated by ViewSolid Biotech Co., Ltd. (Beijing, China). Briefly, CRISPR/Cas9 technology replaced the genomic DNA fragment from intron 2 and 4 of the WDR45 gene with a donor DNA fragment containing LoxP-flanked exon 2 to 4 WDR45. Based on Cas9/gRNA activity screen and target location, high-activity gRNAs (target DNA sequence: GCACAAACACCAAGCATGGGG; ACACAGTGCTATTGGGGCTGG) were selected for microinjection into C57BL/6J fertilized eggs to produce conditional gene knockout mice.
To achieve a mouse model that conditionally knocked out WDR45 in the DAergic system, we bred DAT CreERT2 mice carrying inducible Cre recombinase under the DAT promoter with heterozygous WDR45 Flox/wt mice to obtain WDR45 Flox/Flox /DAT CreERT2 mice. The DAT CreERT2 mouse was kindly gifted by the Günther Schütz group and was generated by recombining a construct containing a modi ed Cre recombinase fused to a modified ligand-binding domain of the estrogen receptor into a bacterial artificial chromosome containing the gene encoding DAT.
All mice were maintained under SPF conditions (temperature, 22 ± 2 °C; air exchange per 20 minutes; 12 h/12 h light/dark cycle with the light on at 6:00 AM) with free access to food and water. Animal care and procedures were carried out per the Laboratory Animal Care Guidelines approved by the Institutional Animal Care Committee at Dalian Medical University. The protocol was approved by the Institutional Animal Care Committee at Dalian Medical University.
To achieve the conditional knockout of WDR45 in the mature DAergic system, TAM (T-5648; Sigma-Aldrich) was employed to treat mice. TAM has dissolved in a corn oil/ethanol (S-5007; Sigma-Aldrich) mixture with a ratio of 10:

Rotarod test
As described previously, mice were trained on the IITC Rotarod (IITC Life Science, Woodland Hills, CA) at 5 r/minutes, twice per day (at 1-hour intervals) for 3 consecutive days, and then on the fourth day, they were tested on the rotating rod with speed auto accelerating from 4 to 40 r/minutes over a period of 5 minutes. The time spent on the rotating rod for each mouse was recorded across three trials at 1-hour intervals. The behavioral assessment was performed at ages 6-8 months, 11-13 months, and 17-19 months.

Y-maze test
The Y-maze test apparatus (Beijing Zhongshidichuang Science and Technology Development Col., Ltd, Beijing, China) was implemented on a white background with three arms (labeled a, b, and c arms) that extended from a central platform at a 120° angle. Each mouse was placed in the center and allowed to freely explore the maze for 6 minutes. The sequence and the total number of arms that the mouse entered were recorded using the observer. An arm entry was successful when the mouse's whole body was within the arm.

Three-chamber social approach test
The three-chamber social approach test was performed as described previously (4). Brie y, a conspeci c mouse of the same sex was placed in a wire-framed steel cage within either the left or right chamber (named novel, the left steel named other), and the subject mouse was allowed to move freely among the three chambers for 5 minutes. A second novel mouse (matched for age and sex) was placed in the remaining wired framed steel cage (named novel, the previous one named familiar), and the subject mouse could move freely for an additional 5 minutes. The cumulative exploration time of the mouse to enter each zone was measured.
Immuno uorescence (IFC) staining Mice were anesthetized with ketamine and perfused transcardially with 40 mL PBS and then 60 mL 4% paraformaldehyde (PFA). After dehydrating 30% sucrose for 72 hours, the brain tissues were cut into 40 μm coronal sections using a Leica cryostat (CM-1950S, Leica, Germany). The slides were incubated with IFC blocking buffer (10% normal goat serum, 1% bovine serum albumin, 0.3% Triton X-100, PBS solution) for 2 hours at room temperature and were then incubated with the primary antibodies overnight at 4 °C (a complete list of primary antibodies information in Table 1). For Ub staining, the sections were subjected to antigen repair using citrate buffer (pH 6.0). The stained sections were visualized and photographed directly with a laser scanning confocal microscope (A1 confocal, Nikon Instruments (Shanghai) Co., Ltd).
The paired images in the figures were collected at the same gain and offset settings. TH-positive cells in the SNc and VTA were calculated in every three sections from bregma −2.80 to −3.64 mm, and the data were collected from 9 slices per animal. The outline of the SNc and VTA was determined according to anatomical landmarks. The analysis of IFC staining on the number of the puncta, axon density, and the mean number of enlarged axon terminals was quantified using ImageJ software.
Quantitative Real-Time PCR (qRT-PCR) Mice were decapitated, and the striatum was isolated on ice. Total RNA was extracted using TRIzol reagent (Invitrogen, Carlsbad, CA, United States), and reverse transcription was performed according to the manufacturer's instructions (638315, Clontech Laboratories, Inc., A Takara Bio Company, United States). qRT-PCR was performed to determine the expression levels of lipid metabolism-related genes using a proper qRT-PCR kit (a complete list of qRT-PCR primers information in Table 2).

Transmission electron microscope (TEM) Analysis
The midbrain SN and striatum were collected rapidly on ice within 3 min into a fixative solution containing 2.5% glutaraldehyde (Servicebio, Wuhan, China) for 2 hours fixtion at room temperature, followed by transfer to 4 degrees for storage. The tissues were washed three times in PBS before postfixing in 1% osmium acid (diluted with 0.1 M PBS solution) at room temperature for 2 hours and were successively dehydrated. After embedding steps, tissues were cut into 80 nm sections using a Leica ultrathin microtome (Leica UC7, Leica, Germany) and stained with 2% uranyl acetate saturated alcohol and lead citrate solution. The stained sections were imaged using TEM (HITACHI, HT7700).
Proteomic Analysis

Sample preparation
The striatal samples were ground into cell powder with liquid nitrogen before being transferred to a 5-mL centrifuge tube. 4 mL lysis buffer (8 M urea, 1% protease inhibitor cocktail) was added to the cell powder, then sonicated three times on ice using a high-intensity ultrasonic processor (Scientz). The remaining debris was removed by centrifugation at 12,000 g at 4 °C for 10 min. Collecting the supernatant and the protein concentration were determined with a BCA kit according to the manufacturer's instructions. For digestion, the protein solution was reduced with 5 mM dithiothreitol for 30 min at 56 °C and alkylated with 11 mM iodoacetamide for 15 min at room temperature in darkness. The protein sample was then diluted by adding 100 mM TEAB to urea concentration less than 2 M. Trypsin was added at 1:50 trypsinto-protein mass ratio for the rst digestion overnight and at 1:100 trypsin-to-protein mass ratio for a second 4 h-digestion. The peptides were desalted by the C18 SPE column.

LC-MS/MS-based proteomic analysis
The tryptic peptides were dissolved in solvent A (0.1% formic acid, 2% acetonitrile/in water) and directly loaded onto a reversed-phase analytical column (25-cm length, 75/100 μm i.d.). Peptides were separated with a gradient from 6% to 24% solvent B (0.1% formic acid in acetonitrile) over 70 min, 24% to 35% in 14 min, and climbing to 80% in 3 min, then holding at 80% for the last 3 min at a constant ow rate of 450 nL/min on a nanoElute UHPLC system (Bruker Daltonics). The peptides were subjected to a capillary source followed by the timsTOF Pro (Bruker Daltonics) mass spectrometry. The electrospray voltage applied was 1.60 kV. Precursors and fragments were analyzed at the TOF detector, with an MS/MS scan range from 100 to 1700 m/z. The timsTOF Pro was operated in parallel accumulation serial fragmentation (PASEF) mode. Precursors with charge state 0 to 5 were selected for fragmentation, and 10 PASEF-MS/MS scans were acquired per cycle. The dynamic exclusion was set to 30 s. The MS/MS data were processed using MaxQuant search engine (v.1.6.15.0). Tandem mass spectra were searched against the human SwissProt database (20422 entries) concatenated with the reverse decoy database. Trypsin/P was speci ed as a cleavage enzyme allowing up to 2 missing cleavages. The mass tolerance for precursor ions was set as 20 ppm in the rst search, and 5 ppm in the main search, and the mass tolerance for fragment ions was set as 0.02 Da. Carbamidomethyl on Cys was speci ed as a xed modi cation, and acetylation on protein N-terminal and oxidation on Met were speci ed as variable modi cations. FDR was adjusted to < 1%.
3. Bioinformatics analysis for proteome GO analysis mainly includes three aspects: 1. Cellular component: Refers to the speci c component of the cell. 2. Molecular function: Mainly describe the chemical activity of the molecule, such as the catalytic activity or binding activity at the molecular level. 3. Biological process: a series of elements in the body that execute a speci c function in order, called the biological process. GO annotation is to annotate and analyze the identi ed proteins with eggnog-mapper software (v2.0). The software is based on the EggNOG database. The latest version is the 5th edition, covering 5,090 organisms (477 eukaryotes, 4445 representative bacteria, and 168 archaebacteria) and 2502 virus genome-wide coding protein sequences. Extracting the GO ID from the results of each protein note and then classifying the protein according to cellular component, molecular function, and biological process. Fisher's exact test is used to analyze the signi cance of GO enrichment of differentially expressed proteins (using the identi ed protein as the background), and p < 0.05 is considered signi cant.

Statistical Analysis
Data are expressed as the means ± SEMs and were analyzed using GraphPad Prism software (version 9.0). Two-way ANOVA followed by Sidak's multiple comparisons test was used for analyses across multiple groups, with Student's t-test used to determine significant differences between the two groups. p < 0.05 is considered significant. All experiments were repeated at least three times, and pilot experiments estimated sample sizes.

Results
Progressive midbrain DAergic neuronal loss in the SN of WDR45 cKO mice To create a mouse model with selective deletion of WDR45 in the midbrain DAergic neurons, we used a TAM-inducible CreERT2/loxp gene-targeting system (Fig. S1a). We generated homozygous WDR45-oxed mice without or with the CreERT2 gene (WDR45 cWT and WDR45 cKO , respectively) and con rmed their genotype using conventional PCR analysis (Fig. S1b). When the mice reached 8 weeks of age, we administered intraperitoneal injections of TAM to both WDR45 cWT and WDR45 cKO mice. Tissues were collected 4 months after TAM administration (when the mice were 6 months old), and we used immuno uorescence (IFC) staining to detect WDR45 protein expression in the tyrosine hydroxylase (TH)labeled DAergic neurons. The results showed a marked decrease in WDR45 expression in the DAergic neurons of WDR45 cKO mice (Fig. S1c), indicating successful deletion of WDR45 in these neurons.
WDR45 cWT and WDR45 cKO mice were subjected to behavioral tests at different ages, including 6-8 months (young), 11-13 months (middle-aged), and 17-19 months (aged). The results showed that the aged WDR45 cKO mice had a signi cant motor impairment, as indicated by a decrease in the total distance traveled in the open-eld test (Fig. S2a) and a notable reduction in stereotypic counts (Fig. S2b), suggesting increased vulnerability to motor activity impairments with aging. However, no abnormalities were observed in the rotarod test (Fig. S2c). In addition to the locomotion de cits, the aged WDR45 cKO mice also showed poor immediate spatial working memory performance, as evidenced by a decreased spontaneous alteration proportion in the Y maze test (Fig. S2d). Furthermore, the 3-chamber social performance test revealed that the aged WDR45 cKO mice displayed a signi cant decrease in social behavior ( Fig. S2e-h), suggesting that WDR45 dysfunction in midbrain DAergic neurons may lead to depression-like behavior in aging mice.
To further investigate the survival of DAergic neurons, we performed IFC staining for TH, a classic marker of DAergic neurons. We analyzed the number of DAergic neurons in the substantia nigra pars compacta (SNc) and ventral tegmental area (VTA) of WDR45 cWT and WDR45 cKO mice at young, middle-aged, and aged stages. We observed a reduction in the number of DAergic neurons in the VTA and SNc of middleaged WDR45 cKO mice compared to age-matched WDR45 cWT mice, and this loss was signi cantly exaggerated in the aged WDR45 cKO mice ( Fig. 1a, b). Additionally, we found a signi cant decrease in the DA content in the SN of aged WDR45 cKO mice compared to that of aged WDR45 cWT mice (Fig. 1c).
We conducted further analysis to investigate the impact of WDR45-de ciency on DAergic neurons at the subcellular level. We used TEM analysis to assess mitochondrial morphology in aged WDR45 cKO mice.
Our results showed the presence of vacuolized mitochondria in the soma of DAergic neurons of aged WDR45 cKO mice but not in age-matched WDR45 cWT mice (Fig. 1d). The perimeter of mitochondria was signi cantly increased in WDR45 cKO mice compared to WDR45 cWT mice, and more than 40% of the mitochondria cristae in the DAergic neurons of WDR45 cKO mice were broken or disappeared (Fig. 1e, f), indicating mitochondrial damage upon WDR45-de ciency in DAergic neurons. We also analyzed the rough endoplasmic reticulum (RER) structure and found a signi cant change in RER morphology in aged WDR45 cKO mice (Fig. 1h). The mean width of RER tubules was signi cantly expanded in the DAergic neurons of WDR45 cKO mice compared to that in WDR45 cWT mice (79.9 nm vs. 51.7 nm) (Fig. 1i). The proportion of RER tubules (> 100 nm) was increased from 6.4% in the DAergic neurons of aged WDR45 cWT mice to 23.9% in the WDR45 cKO mice (Fig. 1j), indicating that the DAergic neurons suffered from severe RER tubular expansion due to WDR45 deletion. Moreover, we found that the numbers of RER and mitochondria detected in the TEM images were dramatically increased in the DAergic neurons of WDR45 cKO mice compared to WDR45 cWT mice (Fig. 1g, k). These ndings suggest that WDR45 deletion may inhibit the clearance or turnover of damaged organelles, accelerating the degeneration of DAergic neurons.
Given that the WDR45 cKO mice experience progressive loss of DAergic neurons during aging, we aimed to investigate the mechanisms underlying cell death. Necroptosis is a regulated form of necrosis and is considered a new mode of cell death. When necroptosis is induced, receptor-interacting protein kinase-3 (RIPK3) becomes activated through phosphorylation and then phosphorylated RIPK3 activates mixed lineage kinase-like (MLKL) through phosphorylation (24,25). To determine if necroptosis was activated in the DAergic neurons of the WDR45 cKO mice, we assessed the presence of phospho-RIPK3 and phospho-MLKL puncta in neuronal cytoplasm using our previously established methods (26). Our data revealed a marked increase in the concentrated puncta of phospho-RIPK3 and phospho-MLKL in the cytoplasm of middle-aged and aged WDR45 cKO mice (Fig. 1l, m, n). These results indicate that necroptosis was activated in the DAergic neurons, leading to progressive DAergic neuronal death in the WDR45 cKO mice during aging.
Axonal degeneration in the striatum of WDR45 cKO mice In addition to the loss of DAergic neurons in the SNc, WDR45 deletion also led to profound nerve ber pathology in the striatum. Our longitudinal study revealed substantial changes in DAergic axonal terminals projected to the striatum of WDR45 cKO mice. Speci cally, we observed signi cant axonal enlargements in the young, middle-aged, and aged WDR45 cKO mice, along with reduced ber density during aging (Fig. 2a-c). It is worth noting that signi cantly more enlargements were accumulated in the nucleus accumbens (NAc), which receives the projection of DAergic neurons in the VTA, than in the caudate putamen (CPu) from SNc DAergic neurons, in the middle-aged and aged WDR45 cKO mice (Fig. 2d, e). These ndings demonstrate that WDR45 cKO mice develop severe DAergic axonal degeneration in the striatum prior to neuronal loss and reveal the differential axonal vulnerability of DAergic neuronal subtypes in response to WDR45 deletion.
Since large enlargements were observed in the axonal terminals, we decided to examine whether the striatal synapses were affected. To study the effect of WDR45 deletion on excitatory synapses of DAergic projections, we conducted TEM analysis for postsynaptic density (PSD), which contributes to information processing and memory formation by changing synaptic strength in response to neural activity (27). The results showed that PSD density was signi cantly reduced (Fig. 2f), and PSD width and average area were signi cantly reduced (Fig. 2g, h). The data suggest that the synaptic structures in the striatum of WDR45 cKO mice have undergone alterations. Furthermore, we assessed the levels of some synaptic proteins in the striatum, including PSD95, a membrane protein of presynaptic vesicles called synaptotagmin 1 (SYT1), a synaptic vesicle protein called synapsin-1 (SYN1), postsynaptic density scaffolding protein called homer scaffold protein 1 (HOMER1), and presynaptic cytomatrix protein bassoon (BSN). We observed a signi cant decrease in the uorescence density of PSD95, SYT1, SYN1, HOMER1, and BSN in the striatum of WDR45 cKO mice (Fig. 2i, j), indicating alterations in synaptic protein levels. These results further support that synaptic signaling transmission is disrupted in WDR45 cKO mice.
Accumulation of increased fragmented tubular ER constitutes a pathological feature of swollen axons in the WDR45 cKO mice Axonal swellings (axonal beadings, bubblings, or spheroids) are hallmarks of degenerating axons, almost universal in neurodegenerative diseases (28, 29). In our study, WDR45 depletion in the DAergic neurons resulted in axonal swellings in the striatum. To gain insights into the molecular basis of axonal degeneration, we evaluated potential candidates by investigating their locations at the axonal enlargements. First, we examined the ER proteins Lysine-Aspartic acid-Glutamic acid-Leucine (KDEL) and climp-63 (30), the tubular ER protein ATL3, and the tubular ER-shaping proteins RTN3 and RTN4 (31,32). RTN4, KDEL, Climp-63, and ATL3 were not observed in the axonal enlargements (Fig. S3). By contrast, RTN3 was highly concentrated at the striatal axonal enlargements in young, middle-aged, and aged WDR45 cKO mice (Fig. 3a, d), suggesting that RTN3 is one of the enlargement components and may contribute to the formation of axonal swellings as an early pathogenic event. Additionally, the density of RTN3-positive puncta at the striatal axons was higher in the older WDR45 cWT mice than in the young WDR45 cWT ones (Fig. 3a), indicating that aging is associated with RTN3 accumulation at the axons. As a typical tubular ER-shaping protein, the accumulation of RTN3 implies that the tubular ER shape may be affected in the striatum. We then investigated the molecular composition of axonal enlargements by determining other tubular ER-shaping proteins, REEP2 and REEP5 (33,34). We found that REEP2 and REEP5 also colocalized with TH-positive enlargements (Fig. 3b, c, e, f), further indicating that the shape of tubular ER in the axons was disrupted upon WDR45 depletion. These ndings highlight the crucial role of ER-shaping proteins in forming axonal enlargements, providing further evidence for the importance of maintaining a normal tubular ER shape in regulating distal axonal homeostasis. The above ndings prompted us to determine whether the tubular ER shape is abnormal in the WDR45 cKO mice. We then examined the tubular ER ultrastructure by TEM in the striatal samples from aged WDR45 cWT and WDR45 cKO mice. Compared to the normally distributed tubular ER in WDR45 cWT mice, a remarkably large accumulation of fragmented tubular ER was noticed in the axons of WDR45 cKO mice ( Fig. 3g-k), supporting the notion that the fragmented tubular ER cluster is a major pathological abnormality associated with axonal degeneration in WDR45 cKO mice.

Disrupted autophagic ux in the DAergic neurons may contribute to the accumulation of tubular ER in axons
To understand what contributes to the accumulation of tubular ER at axons, we rst examined whether autophagic ux was disrupted in the DAergic neurons of WDR45 cKO mice. We stained midbrain sections to detect the expression and distribution of SQSTM1 (p62), ubiquitin (Ub), and LC3, a classical marker for autophagic vesicles. Compared with WDR45 cWT mice, we observed distinct p62-positive puncta accumulated in the soma of DAergic neurons in young WDR45 cKO mice, and this accumulation was aggravated in the aged WDR45 cKO mice (Fig. 4a, b). Additionally, we found that Ub expression was signi cantly increased in the nucleus of DAergic neurons of young WDR45 cKO mice and in the cytoplasm of DAergic neurons of aged WDR45 cKO mice, of which the Ub staining was not entirely colocalized with p62-positive puncta (Fig. 4a, c). Similarly, LC3-positive puncta were also concentrated in the cell body of DAergic neurons in the WDR45 cKO mice (Fig. 4a, d). These data suggest that WDR45 depletion induced an early impairment of autophagic ux in the DAergic neurons, likely triggering axonal and cell body degeneration.
To assess whether LC3-labeled autophagosomes are presented in axonal enlargements, we stained the striatal sections and found that LC3-positive puncta were absent in the TH-positive axonal enlargements (Fig. 4e), indicating that the LC3-labeled autophagosomes did not directly contribute to the formation of axonal enlargements. Furthermore, lysosome marker Lamp1 was also absent in the axonal enlargements (Fig. 4f). The autophagic substrates p62 and Ub were also not colocalized with axonal enlargements (Fig.  4g, h). Therefore, the accumulations of autophagic proteins were mainly observed in the soma but not in the axons of DAergic neurons de cient in WDR45. We speculate that disrupting autophagic ux in the DAergic neurons may lead to axonal enlargements by promoting tubular ER accumulation at axons. To test this hypothesis, we employed another mouse model with damaged autophagic ux, the VMP1 cKO mice that conditionally knocked out autophagic gene VMP1 in the DAergic neurons upon TAM treatment postnatally (26). VMP1 cKO mice also displayed severe damage to autophagic ux and large axonal enlargements in the striatum (26). RTN3, REEP2, and REEP5 were highly accumulated at the TH-positive axonal enlargements in the striatum of 12-month-old VMP1 cKO mice ( Fig. 4i-n), indicating that defective autophagy may induce axonal accumulation of tubular ER. Together, these results suggest that the abnormal clustering of tubular ER in axons may have pathological effects on the brain. Furthermore, our ndings provide additional evidence that autophagy plays a critical role in maintaining axonal homeostasis by regulating the shape and accumulation of tubular ER.
Exploring the proteome landscape of striatum in the WDR45 cKO mice To gain a deeper molecular understanding of how the pathological abnormalities in the DAergic axons affect the striatal cells, we dissected the striatal samples from aged WDR45 cKO mice and age-and sexmatched WDR45 cWT mice for proteomic analysis. Principal component analysis (PCA) revealed the distinct proteome pro les of WDR45 cWT mice and WDR45 cKO mice (Fig. 5a). Further proteomic analyses registered 6,290 targets, of which 162 differentially expressed proteins were identi ed (DEPs; > 1.3-fold change cutoff, p < 0.05). Among the differentially expressed proteins, 115 DEPs were up regulated, and 47 DEPs were downregulated (Fig. 5b, c, and Supplementary Table 1 and cold-inducible RNA binding protein (Cirbp), as well as in regulating anatomical structure morphogenesis, like secreted protein acidic and cysteine-rich (Sparc), angiotensinogen (Agt), and the protein activator of interferon-induced protein kinase EIF2AK2 (Prkra) (Fig. 5d). The top 20 downregulated DEPs are most associated with nitrogen compounds' metabolic process, such as complex integrator subunit 4 (Ints4), keratin 2 (Krt2), and strawberry notch homolog 2 (Sbno2), and proteins with cell morphogenesis, such as protein cordon-bleu (Cobl), amyloid β-A4 precursor protein-binding family B member 1-interacting protein (Apbb1ip) (Fig. 5d).
To support the biological signi cance of these DEPs, we performed Gene Ontology (GO) annotation analysis, which depicts protein functions in three categories: biological processes (BP), cellular components (CC), and molecular functions (MF). The most correlated BP of the DEPs is the regulation of the biological process, metabolic-related process, including organic substance, cellular, primary, and nitrogen compound metabolic process, and that regulation of anatomical structure development (Fig. 5e). Regarding CC, these DEPs are mostly found in the intracellular anatomical structure, cytoplasm, and organelle (Fig. 5e). The analysis results of MF show that these DEPs are primarily associated with protein binding, ion binding, and hydrolase activity (Fig. 5e). To further investigate the functions and signaling pathways of the DEPs, we performed GO enrichment analyses. The top 20 enriched BP pathways are related to amino acids' catabolic and biosynthetic processes, positive regulation of the lipid catabolic process, protein depalmitoylation, etc. Notably, many DEPs are involved in regulating enzymatic activity, like aminomethyltransferase and steroid hydroxylase, that are enriched in the amino acid metabolic process pathway, including lysine, L-cysteine, serine family amino acids, glycine, aspartate family amino acids, and in the tricarboxylic acid metabolic process, lipid catabolic process (Fig. 5f, g).
Overall, the proteomic data indicate that most DEPs are related to lipid and amino acid metabolism, particularly catabolism. This suggests that deleting WDR45 in DAergic neurons leads to signi cant metabolic changes in the striatum, resulting in catabolic reactions in amino acids and lipids. The activated catabolism of these molecules may indicate an energy supply de cit in the striatum, supported by the enrichment of DEPs in the tricarboxylic acid metabolic process that produces adenosine triphosphate (ATP) for cellular energy (Fig. 5f).
The connection of the phospholipid metabolism-related genes with the striatal pathology Tubular ER dynamics are closely related to phospholipid metabolism (19). According to the proteomic data, 18% of up-regulated DEPs and 15% of down-regulated DEPs regulate lipid metabolism (Fig. 6a, b).
To further understand the correlation of striatal pathology with lipids, especially with phospholipids, we analyzed those DEPs related to lipid metabolism by examining the message RNA (mRNA) level using qRT-PCR in both young and aged WDR45 cKO mice. The mRNA level of Lpcat1, a gene that encodes an enzyme that plays a role in phospholipid metabolism, speci cally in the conversion of lysophosphatidylcholine to PC, was signi cantly up-regulated in the striatum of both young and aged WDR45 cKO mice (Fig. 6c, d), indicating that Lpcat1 is involved in the early events following the WDR45 depletion; more importantly, the trend of change in the expression of Lpcat1 in the young and aged mice is consistent, suggesting that Lpcat1 may be one of the initiating factors for striatal axonal pathology. Additionally, we detected the expressions of other genes that participate in lipid metabolism, including Abhd4, Etnppl, apolipoprotein D (APOD), sorting nexin 32 (Snx32), and myelin basic protein (MBP), TIAM Rac1 Associated GEF 2 (Tiam2), Oligophrenin 1 (Ophn1). The results showed that the expressions of Abhd4 and Etnppl, both participate in the regulation of the phospholipid metabolic process, were signi cantly reduced in the striatum of young WDR45 cKO mice compared to that in young WDR45 cWT mice (0.92 vs 0.74, 1.10 vs 0.61, respectively) while markedly increased in the aged WDR45 cKO mice (0.88 vs 1.02, 1.83 vs 3.38, respectively), indicating that the expression of these two genes is simultaneously affected by aging or aging-related processes (Fig. 6c, d). The expression of APOD was signi cantly downregulated in the young WDR45 cKO mice but showed no apparent alteration in the aged WDR45 cKO mice (Fig. 6c, d). Snx32, MBP, Tiam2, and Ophn1 expression showed an up-regulated trend in the young and aged WDR45 cKO mice (Fig. 6c, d). In summary, our ndings emphasize the role of phospholipid biosynthesis and catabolism-regulating molecules in the striatal pathology resulting from WDR45 dysfunction.

Discussion
In this study, we generated and characterized WDR45 cKO mice, which exhibited neurodegeneration of midbrain DAergic neurons, particularly axonal degeneration. We observed the accumulation of abundant fragmented tubular ER in axonal enlargements, impaired autophagic ux in cell bodies, and alterations in DEPs that regulate phospholipid biosynthesis and catabolism. These ndings provide a possible molecular mechanism for axonal degeneration in DAergic neurodegeneration induced by WDR45 de ciency (Fig. 7).
Several neurodegenerative diseases, including Alzheimer's disease, Parkinson's disease, and amyotrophic lateral sclerosis, exhibit apparent defects in axonal transport (35,36). These defects manifest as axonal swellings or spheroids with an aberrant accumulation of axonal cargoes, cytoskeletal proteins, and lipids (37). Prominent axonopathy is induced by the overexpression of pathological proteins involved in several neurodegenerative diseases (37,38 (39). They found enlarged axonal terminals selectively in the mature ATG7-de cient DAergic neurons (39). Similar axonal enlargements were found in the WDR45 cKO mice, VMP1-de cient DAergic neurons (26), and Perry syndrome-associated p150 Glued -de cient DAergic neurons (40). However, the underlying mechanism of how autophagy regulates axonal morphology is still unclear. One study suggests autophagy regulates axonal transmission by controlling axonal ER (20). They found that when ATG5 was deleted from hippocampal neurons, tubular ER was selectively accumulated in axons, followed by ryanodine receptors relative to ER stores in axons, resulting in aberrant calcium release and neurotransmission (20). Moreover, Wan et al. found that WDR45 contributed to neurodegeneration by regulating ER stress and ER quality control, suppressing ER stress, or activating autophagy through mTOR inhibition, alleviating cell death (4). Their observations emphasize the regulation of WDR45 in ER homeostasis through the macroautophagy machinery, which aligns with our hypothesis that axonal swellings in WDR45 cKO mice originate from axonal ER accumulation and that autophagy is responsible for the axonal ER accumulation. Tubular ER is responsible for lipid biogenesis and calcium signaling and provides contact sites for other organelles involved in axonal pathology (19,41). In our study, the morphology of tubular ER in axons is impacted when WDR45 is de cient. Tubular ER becomes fragmented and accumulates in the axons, ultimately promoting axonal enlargement formation. Conversely, sheet ER, which represents rough ER, does not participate in axonal pathology because it is mainly responsible for protein synthesis, processing, and sorting in the soma (41), as con rmed by our nding that the sheet ER markers, KDEL, and climp63, do not colocalize with axonal enlargements in WDR45 cKO mice.
Our results indicate that the morphology of tubular ER is important in maintaining axonal homeostasis, which is supported by the proteomic data showing the strong response of phospholipid metabolism in the striatum to WDR45 depletion. The biosynthesis and catabolism of phospholipids play a fundamental role in the morphology and composition of the ER (16). Lpcat1, an important ER-resident enzyme that catalyzes PC biosynthesis, is substantially up-regulated in the striatum of WDR45 cKO mice, indicating that WDR45 directly or indirectly inhibits Lpcat1 expression and through which WDR45 may participate in the regulation of axonal ER morphology. Previous studies have emphasized the important role of Lpcat1 in lung cancers (42), and recent studies have shown that Lpcat1 also plays a role in neurological diseases. Lpcat1 regulated α-synuclein (αSyn) pathology and cytotoxicity (43). Suppression of Lpcat1 reduces αSyn accumulations and toxicity, and overexpression of Lpcat1 promotes phosphorylated S129 αSyn positive aggregation (43). A phospholipid product of Lpcat1 enzymatic activity, 1,2-dipalmitoyl-snglycero-3-phosphocholine, similarly promotes neuronal αSyn pre-formed bril-seeded aggregation (43).
The relationship between axonal degeneration induced by WDR45 depletion and Lpcat1 expression and associated phospholipid products is an interesting area for future research.
Recent studies indicate phospholipids, and their corresponding compounds are essential for autophagy regulation. The de novo formation of autophagosomes originates from the biosynthesis of phospholipids (44), and the loss of PC biosynthesis compromises the closure of the autophagic membrane and autophagic ux (45). Mitophagy can be inhibited by changing the LC3 residues that carry the cardiolipininteraction sites. Cardiolipin is an analogy to phospholipid found in the inner mitochondrial membrane. It is an elimination signal for mitophagy in neural cells (46). The mutation of Spns1, a protein that acts as a proton-dependent lysophosphatidylcholine (LPC) and lysophosphatidylethanolamine (LPE) transporter, will lead to lysosomal accumulation of LPC and LPE with pathological consequences on lysosomal function (47). Despite these studies supporting the regulation of phospholipids on autophagy, there is currently no report that WDR45 regulates the autophagy process through phospholipids. Our study demonstrates that the deletion of WDR45 causes axonal swellings and fragmented tubular ER accumulation in the swollen axons. To determine the underlying pathways and molecular mechanisms regulating the axonal pathology, we performed proteomic analysis of the striatum from WDR45 cKO mice compared with its littermate WDR45 cWT mice. We found signi cant changes in the expression of phospholipid metabolism-related regulatory proteins, indicating that phospholipid metabolism may contribute to axonal degeneration. To further clarify the regulation of lipid metabolism on tubular ER and its relationship with autophagy will signi cantly improve the mechanistic understanding of axonal degeneration in many neurodegenerative diseases.
Additionally, we screened several pathways by GO enrichment and found that the most activated pathways include the phospholipid metabolic process, amino acids metabolic process, and tricarboxylic acid metabolic process. The defects in these pathways may cause major disturbances in the catabolism of amino acids, lipid catabolism, and tricarboxylic acid metabolism, all related to energy supply. The amino acid metabolism interacts with lipid metabolism in regulating neuronal excitability and survival (48), and these metabolic disturbances are closely related to neurodegeneration (49,50). Due to their anatomical structure, axons are more sensitive to endogenous and exogenous stimuli, such as energy shortages. Mitochondria supply the energy of neurons through oxidative phosphorylation reactions, but given the elongated anatomical structure of the axons, the local ATP supply of the axons is more critical for their survival. Mitochondria are anchored at distal axons and synapses ideally as local energy sources by generating ATP through oxidative phosphorylation (51). Regulating the tra cking and anchoring status of axonal mitochondria ensures that metabolical areas are constantly supplied with ATP. Several pathways and molecules are crucial in regulating mitochondrial transport to meet axonal energy supply and facilitating axonal regeneration, such as AMP-activated protein kinase-p21-activated kinase energy signaling pathway, myosin VI (52), AKT-P21-activated kinase 5 axis (53), syntaphilin (54). Combined with the DEPs in our proteomic data, these pathways provide a clue to study the mechanism of energy supply damage in the axonal degeneration of neurodegenerative diseases.

Conclusions
In conclusion, our research provides insights into the pathological mechanisms underlying WDR45 de ciency-induced axonal degeneration. Our ndings suggest that this degeneration involves abnormal phospholipid metabolism and damaged autophagy. While this study provides a molecular basis for axonal degeneration in BPAN and other neurodegenerative diseases, further mechanistic investigations are necessary to fully understand the complex relationship between tubular ER, autophagy, and phospholipid metabolism in axonal degeneration. The elucidation of such a relationship will facilitate the development of more effective strategies for preventing or reversing axonal degeneration in the context of WDR45 de ciency and related disorders. Tables Table 1 Antibodies used in this study.  Axonal degeneration in the striatum of WDR45 cKO mice. a IFC staining for striatal axons, including the NAc and CPu, was performed using an antibody against TH (red) in young, middle-aged, and aged  Increasing fragmented tubular ER constitutes a pathological feature of axons in WDR45 cKO mice. a IFC analysis for RTN3 in the NAc of WDR45 cWT mice and WDR45 cKO mice was performed using antibodies against RTN3 (green) and TH (red). The nuclei were labeled with DAPI (blue). Scale bar, 10 μm. b IFC staining for REEP2 in the NAc of aged WDR45 cWT mice and WDR45 cKO mice was performed using antibodies against REEP2 (green) and TH (red). The nuclei were labeled with DAPI (blue). Scale bar, 10 μm. c IFC staining for REEP5 in the NAc of aged WDR45 cWT mice and WDR45 cKO mice was performed using antibodies against REEP5 (green) and TH (red). The nuclei were labeled with DAPI (blue). Scale bar, 10 μm. d Analysis of RTN3-and TH-positive enlargements (N= 8-9 slices from 3 mice per genotype). e Analysis of the number of REEP2-and TH-positive enlargements (N= 9 slices from 3 mice per genotype). f Analysis of the number of REEP5-and TH-positive enlargements (N= 9 slices from 3 mice per genotype).
g-j Samples from aged WDR45 cWT mice and WDR45 cKO mice were examined by TEM, and representative TEM images of observed tubular ER at the axons of the striatum are shown. The tubular ER is highlighted in black. Scale bar, 500 nm. For enlarged images, 250 nm. k The mean length of tubular ER was analyzed from aged WDR45 cWT mice and WDR45 cKO mice (N=8-9 slices from 3 mice for each genotype). Data were analyzed by using Student's t-test. Data are represented as the mean±SEM. **** p < 0.0001. The potential interactions of autophagy, tubular ER, and metabolic processes in the axonal pathology of WDR45-de ciency-induced DAergic neurodegeneration. Autophagy is essential for maintaining axonal homeostasis by controlling tubular ER. Phospholipid metabolism interacts with amino acid metabolism and potentially regulates tubular ER. These complex interactions jointly regulate axonal homeostasis.

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